| 摘要: | 紅花 (Carthamus tinctorius) 是傳統中草藥用於治療心血管疾病。在目前的研究中，我們去探討紅花萃取物 (CTF) 和紅花的單一成分（CTF-4-7C-5）對於Lipopolysaccharide (LPS)所引起的心肌細胞株H9c2凋亡是否具有改善或抑制的效果之分子機制研究。首先我們使用LPS誘導傷害細胞H9c2時間為12小時，隨後使用CTF濃度劑量分別為0, 1, 10, 25 μg/ml或CTF-4-7C-5濃度劑量0, 0.1, 0.5, 1, 10μM治療，並24小時後MTT，西方墨點，流式細胞儀， tunel assay，線粒體膜電位檢測， siRNA transfection來探討CTF和CTF-4-7C-5之保護心肌細胞株H9c2以LPS所誘導細胞凋亡的結果。本研究結果發現CTF和CTF-4-7C-5對細胞H9c2有明顯產生生長及提高存活率的作用，隨劑量增加存活率也隨之增加。在細胞凋亡方面，經過LPS處理後，會誘發TUNEL-positive凋亡細胞的增加、TNF-α, FAS-L, FAS, FADD, BID, t-BID, 活化態caspase-3, 8, 9蛋白與促凋亡蛋白的表現，同時也會引起粒線體膜電位的不穩定進而促使細胞色素cytochrome c的釋放。我們還發現，H9c2細胞在LPS處理後，其生存途徑之蛋白下降的趨勢，在加入CTF後有回復的現象，進而達到抵抗LPS所造成細胞凋亡之效果。值得注意的是，使用IGF-IR抑製劑或IGF-IR siRNA進一步的實驗確認CTF透過活化IGF-1R以防止LPS誘導的H9c2細胞凋亡。
綜和上述實驗結果，CTF 和CTF-4-7C-5抑制FAS-death receptor和激活IGF-1R途徑以防止LPS誘導的H9c2細胞凋亡。
Carthamus tinctorius is a traditional Chinese medical herb that has been used as a treatment for cardiovascular disease. The present study was performed to investigate the effect of Carthamus tinctorius extract (CTF) or the CTF pure compound (CTF-4-7C-5), on rat myocardium cell line H9c2 and the possible molecular mechanism. H9c2 cells were treated with LPS (2 μg/ml) for 12 h, and subsequently treated with CTF (1-25 μg/ml) or CTF-4-7C-5 (0.1-1μM). The incubation continued for another 24 h and the cells were analyzed with MTT assay, cell morphological changes assays, western blot analysis, flow cytometry, TUNEL, JC-1 staining and siRNA transfection assays. CTF or CTF-4-7C-5 increased H9c2 cell viability in a dose-dependent manner. Significant increases of TUNEL-positive cells, TNF-α, FAS-L, FAS, FADD, caspase-3, 8, 9 levels, cytosolic cytochrome c, pro-apoptotic protein markers, instability of mitochondria membrane potential, were observed in LPS-treated H9c2 cells. Unexpectedly, these LPS-enhanced effects were dose-dependently reduced by CTF at all doses, and 25 μg/ml was the most effective. It was also found that the survival pathway may involve anti-apoptotic effect of CTF on LPS-treated H9c2 cells. IGF-IR siRNA or inhibitors reversed the CTF effects, confirming that CTF activate IGF-1R to attenuate LPS-induced H9c2 cell apoptosis.
The current findings indicate that CTF and compound CTF-4-7C-5 inhibit FAS-death receptor pathway and activate Insulin-like growth factor-I receptor pathway to reduce LPS-induced H9c2 cardiomyoblast cell apoptosis. |
